The goal of the proposed research is to determine the molecular mechanisms by which enzymes utilize the free energy of ATP hydrolysis to bring about changes in the structure of the DNA helix. The research will focus on the DNA strand pairing reactions that are carried out by DNA recombinases during homologous genetic recombination. In particular, the mechanism of the protypical DNA recombinase, the recA protein of Escherichia coli, will be analyzed. Detailed mechanistic models will be developed for the various elementary reactions of the recA protein that together constitute a recA protein-promoted strand invasion event. In addition, site-directed mutagenesis will be used, in combination with sequence analysis and chemical modification, to create new mutant recA proteins that will be used to test these mechanistic models. These mutant proteins will also be analyzed in the more complex strand exchange reactions in order to determine how specific well-defined defects in the various elementary reactions of the recA protein affect the overall strand exchange process. These studies will lead to an understanding of the mechanistic principles that are involved in an ATP-dependent DNA recombinase- promoted strand pairing event.